1. Field of the Invention
The invention relates to the therapeutic use of oligonucleotides, both in the antisense approach, and as immunostimulatory agents.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modem molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modem approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23:3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Antl. Acad. Sci. USA 85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
More recently, several researchers have demonstrated the validity of the antisense approach to therapeutic treatment of disease. Crooke, Antisense Nucleic Acid Drug Dev. 8: vii-viii, discloses the successful marketing approval of a phosphorothioate oligonucleotide for the treatment of human cytomegalovirus-induced retinitis. Unfortunately, the use of phosphorothioate oligonucleotides has become more complex than originally expected. Certain effects caused by phosphorothioate oligonucleotides could not be explained by the expected antisense mechanism. For example, McIntyre et al., Antisense Res. Dev. 3: 309-322 (1993) teaches that a xe2x80x9csensexe2x80x9d phosphorothioate oligonucleotide causes specific immune stimulation. This and other side effects have complicated the picture for phosphorothioate oligonucleotides.
On the other hand, the observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in developing this side effect as a therapeutic tool. These efforts have focussed on phosphorothioate oligonucleotides containing the dinucleotide CpG. Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity. Krieg et al., Nature 371: 546-549 (1995) discloses that phosphorothioate CpG-containing oligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest. 98: 1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu et al., Vaccine 16: 1216-124 (1998) teaches that CpG-containing phosphorothioate oligonucleotides enhance immune response against influenza virus. McCluskie and Davis, The Journal of Immunology 161: 4463-4466 (1998) teaches that CpG-containing oligonucleotides act as potent adjuvants, enhancing immune response against hepatitis B surface antigen.
These reports make clear that there is a need to be able to modulate the immune response caused by CpG-containing oligonucleotides. Ideally, such modulation should include decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications.
The invention provides methods for modulating the immune response caused by CpG-containing oligonucleotides. The methods according to the invention enables both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications. Thus, the invention further provides oligonucleotides having optimal levels of immunostimulatory effect for either application and methods for making and using such oligonucleotides.
The present inventor has surprisingly discovered that positional modification of CpG-containing oligonucleotides dramatically affects their immunostimulatory capabilities. In particular, 3xe2x80x2 alkylation or alkoxylation of oligonucleotides, or introduction of an uncharged internucleoside linkage, at particular positions 5xe2x80x2 or 3xe2x80x2 to the CpG dinucleotide either enhances or reduces their immunostimulatory effect in a reproducible and predictable manner.
In a first aspect, the invention provides a method for reducing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing a 3xe2x80x2 substituted nucleoside into the oligognucleotide at a position adjacent to, and on the 5xe2x80x2 and/or 3xe2x80x2 side of the CpG dinucleotide. In preferred embodiments, this method includes creating a 2xe2x80x2-5xe2x80x2 linkage between the 2xe2x80x2 position of a 3xe2x80x2 substituted nucleoside and the 5xe2x80x2 position of another nucleoside, which may or may not be a 3xe2x80x2 substituted nucleoside.
In a second aspect, the invention provides a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 3xe2x80x2 substituted nucleoside at a position adjacent to, and on the 5xe2x80x2 and/or 3xe2x80x2 side of the CpG dinucleotide. In preferred embodiments, CpG-containing oligonucleotides according to this aspect of the invention include a 2xe2x80x2-5xe2x80x2 linkage between the 2xe2x80x2 position of a 3xe2x80x2 substituted nucleoside and the 5xe2x80x2 position of another nucleoside, which may or may not be a 3xe2x80x2 substituted nucleoside.
In a third aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, the method comprising administering to the mammal a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 3xe2x80x2 substituted nucleoside at a position adjacent to, and on the 5xe2x80x2 and/or 3xe2x80x2 side of the CpG dinucleotide. In preferred embodiments, CpG-containing oligonucleotides used in this aspect of the invention include a 2xe2x80x2-5xe2x80x2 linkage between the 2xe2x80x2 position of a 3xe2x80x2 substituted nucleoside and the 5xe2x80x2 position of another nucleoside, which may or may not be a 3xe2x80x2 substituted nucleoside.
In a fourth aspect, the invention provides a method for reducing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing an uncharged internucleoside linkage into the oligonucleotide at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide.
In an fifth aspect, the invention provides a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises an uncharged internucleoside linkage at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide.
In a sixth aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, the method comprising administering to the mammal a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises an uncharged internucleoside linkage at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide.
In a seventh aspect, the invention provides a method for reducing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing a 2xe2x80x2-5xe2x80x2 internucleoside linkage into the oligonucleotide at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide.
In an eighth aspect, the invention provides a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 2xe2x80x2-5xe2x80x2 internucleoside linkage at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide.
In a ninth aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, the method comprising administering to the mammal a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 2xe2x80x2-5xe2x80x2 internucleoside linkage at a position adjacent to, and on the 5xe2x80x2 side of the CpG dinucleotide.
In a tenth aspect, the invention provides a method for increasing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing into the oligonucleotide a 3xe2x80x2 substituted nucleoside at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide. In preferred embodiments, this method includes creating a 2xe2x80x2-5xe2x80x2 linkage between the 2xe2x80x2 position of a 3xe2x80x2 substituted nucleoside and the 5xe2x80x2 position of another nucleoside, which may or may not be a 3xe2x80x2 substituted nucleoside.
In a eleventh aspect, the invention provides CpG-containing oligonucleotides having increased immunostimulatory effects, the oligonucleotide comprising a 3xe2x80x2 substituted nucleoside at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain embodiments, the oligonucleotide is not an antisense oligonucleotide. In preferred embodiments, CpG-containing oligonucleotides according to this aspect of the invention include a 2xe2x80x2-5xe2x80x2 linkage between the 2xe2x80x2 position of a 3xe2x80x2 substituted nucleoside and the 5xe2x80x2 position of another nucleoside, which may or may not be a 3xe2x80x2 substituted nucleoside.
In an twelfth aspect, the invention provides a method for inducing an immune response in a mammal, the method comprising administering to the mammal an oligonucleotide comprising a 3xe2x80x2-substituted nucleoside at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CPG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide. In preferred embodiments, CpG-containing oligonucleotides used in this aspect of the invention include a 2xe2x80x2-5xe2x80x2 linkage between the 2xe2x80x2 position of a 3xe2x80x2 substituted nucleoside and the 5xe2x80x2 position of another nucleoside, which may or may not be a 3xe2x80x2 substituted nucleoside.
In a thirteenth aspect, the invention provides a method for increasing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing into the oligonucleotide an uncharged internucleoside linkage at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide.
In a fourteenth aspect, the invention provides CpG-containing oligonucleotides having increased immunostimulatory effects, the oligonucleotide comprising an uncharged internucleoside at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof.
In a fifteenth aspect, the invention provides a method for inducing an immune response in a mammal, the method comprising administering to the mammal an oligonucleotide comprising an uncharged internucleoside linkage at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide.
In a sixteenth aspect, the invention provides a method for increasing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing into the oligonucleotide a 2xe2x80x2-5xe2x80x2 internucleoside linkage at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide.
In a seventeenth aspect, the invention provides CpG-containing oligonucleotides having increased immunostimulatory effects, the oligonucleotide comprising a 2xe2x80x2-5xe2x80x2 internucleoside linkage at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof.
In an eighteenth aspect, the invention provides a method for inducing an immune response in a mammal, the method comprising administering to the mammal an oligonucleotide comprising a 2xe2x80x2-5xe2x80x2 internucleoside linkage at a position selected from the group consisting of 3rd nucleoside 5xe2x80x2 to the CpG dinucleotide, 4th nucleoside 5xe2x80x2 to the CpG dinucleotide, 5th nucleoside 5xe2x80x2 to the CpG dinucleotide, 6th nucleoside 5xe2x80x2 to the CpG dinucleotide, 2nd nucleoside 3xe2x80x2 to the CpG dinucleotide, 3rd nucleoside 3xe2x80x2 to the CpG dinucleotide, 4th nucleoside 3xe2x80x2 to the CpG dinucleotide, 5th nucleoside 3xe2x80x2 to the CpG dinucleotide, 6th nucleoside 3xe2x80x2 to the CpG dinucleotide, 7th nucleoside 3xe2x80x2 to the CpG dinucleotide, 8th nucleoside 3xe2x80x2 to the CpG dinucleotide, 9th nucleoside 3xe2x80x2 to the CpG dinucleotide, 10th nucleoside 3xe2x80x2 to the CpG dinucleotide, and combinations thereof. In certain preferred embodiments, the oligonucleotide is not an antisense oligonucleotide.